Formulations of water insoluble or poorly water soluble drugs in lipidated glycosaminoglycan particles and their use for diagnostics and therapy

ABSTRACT

The invention provides a formulation of water insoluble or poorly water soluble drugs encapsulated in lipidated glycosaminoglycan particles for targeted drug delivery.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of application Ser. No.11/718,485, filed Apr. 21, 2008, which is a 371 national stage ofinternational application no. PCT/US2005/039224, filed Nov. 2, 2005,which claims the benefit of priority under 35 U.S.C. §119(e) fromprovisional U.S. application No. 60/623,862, filed Nov. 2, 2004, theentire content of which is herein incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to drug delivery and formulation andspecifically to particles of lipidated glycosaminoglycans encapsulatingwater insoluble or poorly water soluble drugs and their use indiagnosing and treating pathological conditions.

2. Description of the Related Art

Glycosaminoglycans, or mucopolysaccharides, along with collagen, are thechief structural elements of all connective tissues. Glycosaminoglycans,or gags, are large complexes of polysaccharide chains associated with asmall amount of protein. These compounds have the ability to bind largeamounts of water, thereby producing a gel-like matrix that forms thebody's connective tissues. Gags are long chains composed of repeatingdisaccharide units (aminosugar-acidic sugar repeating units). Theaminosugar is typically glucosamine or galactosamine. The aminosugar canalso be sulfated. The acidic sugar may be D-glucuronic acid orL-iduronic acid. In vivo, gags other than hyaluronic acid are covalentlybound to a protein, forming proteoglycan monomers. The polysaccharidechains are elongated by the sequential addition of acidic sugars andaminosugars.

Among the most common gags are hyaluronic acid, keratan sulfate,chondroitin sulfate, heparin sulfate, and dermatin sulfate. Gags may bechemically modified to contain more sulfur groups than in theirinitially extracted form. In addition, gags may be partially orcompletely synthesized and may be of either plant or animal origin.

Hyaluronic acid is a naturally occurring member of the glycosaminoglycanfamily which is present in particularly high concentration in thecartilage and synovial fluid of articular joints, as well as in vitreoushumor, in blood vessel walls, and umbilical cord and other connectivetissues. Hyaluronic acid can be in a free form, such as in synovialfluid, and in an attached form, such as an extracellular matrixcomponent. This polysaccharide consists of alternatingN-acetyl-D-glucosamine and D-glucuronic acid residues joined byalternating β-1,3-glucuronidic and β-1,4-glucosaminidic bonds. In water,hyaluronic acid dissolves to form a highly viscous fluid. The molecularweight of hyaluronic acid isolated from natural sources generally fallswithin the range of 5×10⁴ up to 10⁷ daltons. Hyaluronic acid has a highaffinity for the extracellular matrix and to a variety of tumors,including those of the breast, brain, lung, skin, and other organs andtissues.

Drug delivery systems are used for maintaining a constant blood level ofa drug over a long period of time by administering a drug into the body,or for maintaining an optimal concentration of a drug in a specifictarget organ by systemic or local administration, and over a prolongedperiod of time. For instance, chemically modified hyaluronic acid can beused for controlled release drug delivery. Balazs et al, in U.S. Pat.No. 4,582,865, reported that cross-linked gels of hyaluronic acid canslow down the release of a low molecular weight substance dispersedtherein but not covalently attached to the gel macromolecular matrix.Other forms of pharmaceutical preparations/formulations are used as drugdelivery systems, including the use of a thin membrane of a polymer orthe use of a liposome as a carrier for a drug.

There are two basic classes of drug carriers: particulate systems, suchas cells, microspheres, viral envelopes, and liposomes; andnon-particulate systems, which are usually soluble systems, consistingof macromolecules such as proteins or synthetic polymers.

The majority of drug dosage forms available in the clinic (over 99%) arehowever formulations of free drugs. Nevertheless, microscopic andsubmicroscopic particulate carriers, performing as drug deliverysystems, are used to improve clinical outcomes compared to treatmentwith free drug. Enclosure within a carrier protects the drug from thebiological environment, reducing the risk of degradation andinactivation. Encapsulation also protects the biological environmentfrom indiscriminate distribution of free drug, reducing the risk oftoxicity and adverse side effects. Carrier mediation reduces pre-maturedrug clearance and ensures a constant blood level of drug and/or anoptimal concentration of drug in target organs over a prolonged periodof time by systemic or by local administration. Particulate carriersperform as sustained-release or controlled-release drug depots, therebycontributing to improved drug efficacy and allowing reduction in dosingfrequency.

Despite the advantages offered, there are some difficulties associatedwith using drug encapsulating biopolymers. For example, biopolymersstructured as microparticulates or nanoparticulates have limitedtargeting abilities, limited retention and stability in circulation,potential toxicity upon chronic administration, and the inability toextravasate. Numerous attempts have been made to bind differentrecognizing substances, including antibodies, glycoproteins, andlectins, to particulate systems, such as liposomes, microspheres, andothers, in order to confer upon them some measure of targeting. Althoughbonding of these recognizing agents to the particulate system has metwith success, the resulting modified particulate systems did not performas hoped, particularly in vivo.

Other difficulties have also arisen when using such recognizingsubstances. For example, antibodies can be patient-specific, and therebyadd cost to the drug therapy. Additionally, not all binding betweenrecognizing substrate and carrier is covalent. Covalent bonding isessential, as non-covalent binding might result in dissociation of therecognizing substances from the particulate system at the site ofadministration, due to competition between the particulate system andthe recognition counterparts to the target site for the recognizingsubstance. Upon such dissociation, the administered modified particulatesystem can revert to a regular particulate system, thereby defeating thepurpose of administration of the modified particulate system.

When it comes to drugs that have poor aqueous solubility (to be referredhenceforth as poorly water-soluble and water insoluble drugs), there isfurther deficiencies in treatment with the free drug. In order togenerate a dosage form that will allow treatment at all, it is necessaryto formulate the water insoluble or poorly water soluble drug in avehicle that will be hydrophobic enough to solubilize the drug, yet behydrophilic enough to accommodate administration into an aqueous medium.These vehicles are usually detergent-like, such as the 1:1 blend ofCremophor EL (polyethoxylated caster oil) and ethanol used forpaclitaxel. The drawback is that these vehicles and other similardetergent-based vehicles are highly toxic and cause hypersensitivityreaction and release of histamines in patients.

U.S. Pat. No. 5,733,892 to Sakurai et al. discloses lipidatedglycosaminoglycan molecules which are soluble in aqueous solution. WO03/015755 discloses a similar system of lipidated glycosaminoglycanparticles which form suspensions of particles in an aqueous phase. Thepresent invention is an improvement of the lipidated glycosaminoglycanparticles of WO 03/015755 as none of the currently available deliverytechnologies provide a satisfactory solution to the problems associatedwith targeted delivery of water insoluble and poorly water solubledrugs.

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

SUMMARY OF THE INVENTION

It is an object of the present invention is to overcome the deficienciesin the prior art.

Another object of the present invention to form lipidatedglycosaminoglycan particles for encapsulating water insoluble or poorlywater soluble drugs.

A further object of the present invention is to deliver such waterinsoluble or poorly water soluble drugs encapsulated in a lipidatedglycosaminoglycan particle.

The present invention provides a formulation of water insoluble orpoorly water soluble drugs encapsulated in lipidated glycosaminoglycanparticles, also termed “gagomers”. Such gagomers are bioadhesivebiopolymers produced by cross-linking a lipid having a primary aminogroup to a carboxylic acid-containing glycosaminoglycan. Microparticlesor nanoparticles are formed in a controlled manner with dominantparticle diameter ranges of about 2-5 microns for microparticles andabout 50-200 nanometers for nanoparticles. Small or large activeingredients/drugs which are water insoluble or poorly water soluble canbe encapsulated/entrapped in these gagomer particles with highefficiency greater than 50%, and usually greater than 80%.

The present invention also provides a pharmaceutical compositioncontaining a water insoluble or poorly water soluble drug/activeingredient encapsulated in lipidated glycosaminoglycan particles.

Other aspects of the present invention include a method for preparingthe lipidated glycosaminoglycan particle encapsulated drug/activeingredient and a method for treating a subject suffering from apathological condition by administering an effective amount of the waterinsoluble or poorly water soluble active ingredient/drug encapsulated inlipidated glycosaminoglycan particles.

A still further aspect of the present invention is directed to animproved method for treating an indication with a water insoluble orpoorly water soluble drug that is effective for treating the indication,where the improvement is that the water insoluble or poorlywater-soluble drug is administered encapsulated in lipidatedglycosaminoglycan particles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the efficiency of paclitaxel encapsulation ingagomers, in the via-DMSO method, as a function of gagomer concentration(in units of mg PE/ml). The points are the experimental data obtained atdifferent initial drug concentration as listed. The solid line and theerror bars are the average encapsulation efficiency and sd for all datapoints.

FIG. 2 is a graph showing the efficiency of paclitaxel encapsulation ingagomers, in the via-ethanol method, as a function of gagomerconcentration (in units of mg PE/ml). The points are the experimentaldata obtained at different initial drug concentration as listed. Thesolid lines are non-theoretical, drawn to emphasize the trends of thedata.

FIG. 3 is a graph showing the efficiency of paclitaxel encapsulation ingagomers, carrier preparation and drug encapsulation in the via-PEmethod, as a function of gagomer concentration (in units of mg PE/ml).The points are the experimental data obtained at different initial drugconcentration as listed. The solid line and the error bars are theaverage encapsulation efficiency and sd, for all data points.

FIG. 4 is a graph showing comparisons of the efficiency of paclitaxelencapsulation in the three types of formulations (i.e., via-DMSO,via-ethanol and via-PE) as a function of the drug/gagomer ratio.Abbreviations used: TX—paclitaxel; GAG—gagomers; HA—hyaluronan;PE—phosphatidylethanolamine. The points are experimental, with thesymbols as follows: open box: via DMSO; open circle: via ethanol; solidcircle: via PE. The solid curve is non-theoretical, drawn to emphasizethe trend of the data.

FIG. 5 is a graph showing the stability of drug-free gagomers and ofpaclitaxel-encapsulating gagomers (prepared via the ethanol method) in50% human serum and at 37° C. as a function of time. Separatelymonitoring HA concentration in gagomers, PE concentration in gagomers,level of drug encapsulation. Drug-free and paclitaxel-encapsulatinggagomers were (TX-GAG) prepared by the via-ethanol method. The pointsare experimental and the specific symbols for each tested variable arelisted. The solid curves are non-theoretical, drawn to emphasize thetrends of the data.

FIG. 6 is a graph showing the kinetics of paclitaxel efflux fromgagomers prepared with the via ethanol method, in PBS and, separately,in 50% serum, under unidirectional flux (sink) conditions. The dependantvariable f_(t) is fraction of total drug released from the formulationat time=t. The points are the experimental data. The solid curves arethe theoretical expectations drawn according to equation (1) and thekinetic parameters listed in the footnotes of Table 3.

FIG. 7 shows a scheme of the production process for the insoluble drugsloaded inside gagomers.

FIG. 8 is a graph showing retention of paclitaxel (TX) in centrifugedpellet washed with phosphate-buffered saline alone (light bars), or thesame buffer but also containing 0.2% bovine serum albumin (dark bars).

FIG. 9 shows differential scanning calorimetry of drug-loaded anddrug-free gagomers, as well as free drug. Upper part: freepaclitaxel—crystalline (dotted line) and hydrated (dashed line) and ofdrug-free gagomers (solid black line). Lower part: mixtures of freecrystalline (solid black line) or hydrated (dashed line) paclitaxel withdrug-free gagomers, at drug:lipid mole ratios of 1:2. Paclitaxel-loadedgagomers (dotted line) also at the drug:lipid mole ratio of 1:2.

FIG. 10 is a graph showing cytotoxicity of free paclitaxel and ofpaclitaxel-loaded gagomers, before and after sterilization, in culturesof B16F10.9 cells.

DETAILED DESCRIPTION OF THE INVENTION

Lipidated glycosaminoglycan particles, also termed “gagomers”, are anovel drug delivery technology for water insoluble and poorly watersoluble drugs/active ingredients that overcomes limitations anddeficiencies of the prior art. This technology provides a versatile,multi-product drug delivery system with marked performance improvementsin terms of both manufacturing processes and clinical outcomes. Thesegagomers have the ability to perform as site-adherent, site-retained,sustained release drug depots for systemic, topical, and regionaladministration. The introduction of this technology for use with waterinsoluble or poorly water soluble drugs is expected to significantlyadvance the state-of-the-art in targeted drug delivery modalities.

Gagomer particles are bioadhesive biopolymers prepared by reacting aglycosaminoglycan with at least one lipid, preferably a phospholipidsuch as phosphatidylethanolamine (PE), more preferably dilaurylphosphatidylethanolamine (DLPE) or dipalmitoyl phosphatidylethanolamine(DPPE) which differ in chain length, to crosslink the carboxylic acidgroups in the glycosaminoglycan with a primary amine in the lipid.Preferably, a coupling agent of the carbodiimide type that forms acovalent bond between carboxyl residues of the glycosaminoglycan and theprimary amine of the lipid is used for the crosslinking.

A unique feature of the gagomer technology discovered by the presentinventors is that these carrier particles, by virtue of their internallipid regions, provide an environment for solublization andencapsulation of water insoluble and poorly water soluble drugs withoutthe need to include any of the toxic and adverse side effect-causingdetergent-like vehicles. Gagomer particles therefore have theadvantageous ability to perform as a targeted delivery system for waterinsoluble and poorly water soluble drugs. The water insoluble and poorlywater soluble drugs are encapsulated in gagomer particles with highefficiency to form the drug encapsulating gagomer particles according tothe present invention. The resultant formulations thus perform assustained release drug depots which are stable in serum and retain drugactivity at levels similar to or better than equivalent doses of freedrug.

The present invention is directed to formulations of water insoluble orpoorly water soluble drugs in gagomer particles, to pharmaceuticalcompositions containing the drug encapsulating gagomers, and to methodsof preparation and use thereof.

A preferred embodiment of a poorly water soluble drug encapsulated inthe gagomer particles according to the present invention is paclitaxel(taxol; TX), a cytotoxic drug that was first isolated from the bark ofthe pacific yew plant. Pacitaxel promotes the creation of intracellularmicrotubulins that are highly stable and dysfunctional, leading to celldeath since normal tubule dynamics are disrupted, thereby prohibitingcell division. Based on this activity, paclitaxel is used as achemotherapeutic agent for a wide variety of cancers, including ovarian,breast, colon, head, non-small cell lung carcinomas, and AIDS associatedKaposi sarcoma. Therapy with paclitaxel faces, as discussed above ingeneral terms, two major problems: (1) it is in dire need of atumor-targeted carrier as with any chemotherapeutic drug; and (2) it hasextremely poor solubility in aqueous solutions. In the current approvedformulations for paclitaxel used in the clinic, pacitaxel is dissolvedin the highly toxic 1:1 blend of Cremophor EL (polyethoxylated castoroil) and ethanol.

Ongoing efforts in the field focus mostly on replacing the CremophorEL/ethanol blend with more favorable vehicles, including carriers suchas PEGylated liposomes. To date however, none has proved to besufficiently satisfactory. While a better vehicle may be useful forovercoming the solubility problem, none of the efforts addresses theissue of tumor targeting. By contrast, the preferred embodiment ofpaclitaxel encapsulated in the gagomer particles according to thepresent invention addresses both the targeting and solubility issues ina single carrier/delivery technology. This should reduce toxicity andadverse effects in patients and at the same time enhance treatmentefficacy, resulting in significant improvements in clinical outcomes.

It has been previously found that water soluble drugs encapsulated inlipidated glycosaminoglycan particles were much more effective than thefree drugs, particularly for cancer cells that have become drugresistant. It appears that the gagomers attach to the cancer cells andthus become depots of drugs which can enter the cells more quickly thanthey are excreted. These water soluble drugs thus have a toxic effect oncells despite the drug-resistance mechanisms that have been developed incancer cells. It is expected that water insoluble or poorly waterinsoluble drugs encapsulated in lipidated glycosaminoglycan particlesaccording to the present invention would also have an enhanced effect oncells compared to the free drug.

In addition to paclitaxel, other non-limiting examples ofdrugs/drug-models with poor aqueous solubility for encapsulation ingagomer particles are presented in Table 1 along with their therapeuticindications. A drug with an aqueous solubility of ≦30 μg/ml isconsidered to be poorly water soluble or water insoluble. For purposesof the present invention, the term “drug” is intended to mean any agentwhich can affect the body therapeutically. Examples of therapeutic drugsinclude chemotherapeutics for cancer treatment, antibiotics for treatinginfections, antifungals for treating fungal infections,anti-inflammatories for treating inflammatory conditions, etc. As shownin the Examples hereinbelow, the lipid dilauryl phosphatidylethanolamineis preferred for encapsulating paclitaxel based on preparation,encapsulation efficiency and cytotoxicty. However, for some other drugs,it may turn out that dipalmitoyl phosphatidylethanolamine would bebetter and more preferred.

TABLE 1 Drugs/drug-models with poor aqueous solubility and therapeuticindications Water insoluble or poorly water soluble drug Therapeuticindication Pacitaxel (Taxol; TX) Cancer/Oncology Nile red (fluorescentprobe) Research tool Etoposide (VP-16, Vepesid) Cancer/OncologyCisplatin Cancer/Oncology Fluorouracil (5-FU) Cancer/OncologyCyclosporin A Transplantion/Immunosuppressant IndomethacinAntiinflammatory Dexamethasone Antiinflammatory Nifedipine Cardiacagents Amphotericin B Antibiotics Antimicrobial Antifungi NeostatinAntibiotics Antimicrobial Antifungi Bethamethasone Steroids CortisoneSteroids

The gagomers used in the present invention are non-toxicmicroparticulate and nanoparticulate drug delivery systems, alsoreferred to as MDDS and NDDS, respectively, that employ drug entrappingadhesive biopolymers. These carriers, when loaded with water insolubleor poorly water soluble drugs, improve clinical outcomes compared to thesame drugs administered in their free form. Moreover, these gagomerparticles have a number of other advantages over other particulatecarriers, such as (1) good and sufficient retention in thecirculation—the glycosaminoglycan component already has the hydrophilicouter shell found to delay opsonization and uptake by the RES, and (2)the bioadhesive nature of the glycosaminoglycan component endows thegagomer particles with the ability to adhere with high affinity to invivo recognition sites and confers a measure of active targeting.

The drug-encapsulating gagomers of the present invention can be used ina pharmaceutical composition to treat a pathological condition in asubject in need thereof. The term “subject” as used herein is taken toinclude humans and other mammals such as cattle, sheep, pigs, goats,dogs, cats, rats, mice, etc., as well as animals including amphibians,birds, reptiles and fish.

Pathological conditions suitable for treatment with the drugencapsulated gagmomers of the present invention include any indicationfor which a water insoluble or poorly water soluble drug is used fortreatment. Examples include, but are not limited to, cancer, bacterialand fungal infections including those secondary to trauma such as burns,infections caused by parasites or viruses, wound healing, inflammation,etc. Thus, the present invention also provides a method for treating asubject suffering from a pathological condition which involvesadministering to the subject an effective amount of the water insolubleor poorly water soluble drug encapsulated in the gagomer according tothe present invention to treat the pathological condition. In the caseof the preferred drug embodiment of pacilitaxel, the pathologicalcondition is cancer.

The present invention is furthermore an improvement over current methodsfor delivering to a subject in need of treatment for a particularindication a water insoluble or poorly water soluble drug that iseffective for treating that indication, the improvement being that thedrug administered to the subject is encapsulated in gagomer particles.

Although naturally-occurring glycosaminoglycans are preferred in thegagomers used in the present invention in order to avoid problems withimmunogenicity and toxicity, synthetic glycosaminoglycans can also beused, as well as natural, synthetic, or semisynthetic molecules,including but not limited to chondroitin, hyaluronic acid, glucuronicacid, iduronic acid, keratan sulfate, heparan sulfate, dermatin sulfate,and fragments, salts, and mixtures thereof. The term “glycosaminoglycan”as used herein further encompasses salts and free acids ofglycosaminoglycan as well as glycosaminoglycans that have beenchemically altered (but not partially hydrolyzed), yet retain theirfunction. These modifications include, but are not limited to,esterification, sulfation, polysulfation, and methylation. Usinghyaluronic acid (HA) as an example, its hyaluronate salts include sodiumhyaluronate, potassium hyaluronate, magnesium hyaluronate, and calciumhyaluronate.

Natural sources of glycosaminoglycans include both plant and animalsources, i.e., beechwood trees and forms of animal cartilage, includingshark cartilage, bovine trachea, whale septum, porcine nostrils, andmollusks such as Perna canaliculus and sea cucumber.

The glycosaminoglycans are used at sizes obtained when they are purifiedfrom their biological sources, and that have not been subjected tochemical and/or biological degradation. For example, for hyaluronicacid, this corresponds to a range of about 1×10⁵ to about 1×10⁷ daltons.

Pharmaceutical compositions containing the drug encapsulating gagomersaccording to the present invention can be administered by any convenientroute, including parenteral, e.g., subcutaneous, intravenous, topical,intramuscular, intraperitoneal, transdermal, rectal, vaginal, intranasalor intraocular. Alternatively or concomitantly, administration may be bythe oral route.

Parenteral administration can be by bolus injection or by gradualperfusion over time. Parenteral administration is generallycharacterized by injection, most typically subcutaneous, intramuscularor intravenous.

Topical formulations composed of the drug encapsulating gagomerparticles of the present invention, penetration enhancers, and otherbiologically active drugs or medicaments may be applied in many ways. Aliquid formation can be applied dropwise, from a suitable deliverydevice, to the appropriate area of skin or diseased skin or mucousmembranes and rubbed in by hand or simply allowed to air dry. A suitablegelling agent can be added to the liquid formulation and the preparationcan be applied to the appropriate area and rubbed in. For administrationto wounds or burns, the gagomers may be incorporated into dosage formssuch as oils, emulsions, and the like. Such preparations may be applieddirectly to the affected area in the form of lotions, creams, pastes,ointments, and the like.

Alternatively, the topical liquid formulation can be placed into a spraydevice and be delivered as a spray. This type of drug delivery device isparticularly well suited for application to large areas of skin affectedby dermal pathologies, to highly sensitive skin or to the nasal or oralcavities. Optionally, the gagomers may be administered in the form of anointment or transdermal patch.

Oral routes of administration are understood to include buccal andsublingual routes of administration.

The gagomers of the present invention may also be administered by otherroutes which optimize uptake by the mucosa. For example, vaginal(especially in the case of treating vaginal pathologies), rectal andintranasal routes are the preferred routes of administration.Furthermore, the gagomers are particularly suited for delivery throughmucosal tissue or epithelia. If administered intranasally, the gagomerswill typically be administered in an aerosol form, or in the form ofdrops. This may be especially useful for treating lung pathologies.Suitable formulations can be found in Remington's PharmaceuticalSciences, 16th and 18th Eds., Mack Publishing, Easton, Pa. (1980 and1990), and Introduction to Pharmaceutical Dosage Forms, 4th Edition, Lea& Febiger, Philadelphia (1985), each of which is incorporated herein byreference.

Depending on the intended mode of administration, the compositions usedmay be in the form of solid, semi-solid or liquid dosage forms, such asfor example, tablets, suppositories, pills, capsules, powders, liquids,suspensions, or the like, preferably in unit dosage forms suitable forsingle administration of precise dosages. The pharmaceuticalcompositions contains the drug encapsulating gagomer particles of thepresent invention and a pharmaceutically acceptable diluent, carrier,excipient, adjuvant, or auxiliary agent. It is preferred that thepharmaceutically acceptable carrier be one which is chemically inert tothe active compounds and which has no detrimental side effects ortoxicity under the conditions of use. The choice of carrier isdetermined partly by the particular active ingredient, as well as by theparticular method used to administer the composition. Accordingly, thereare a wide variety of suitable formulations of the pharmaceuticalcompositions of the present invention.

Suitable excipients are, in particular, fillers such as saccharides(e.g., lactose or sucrose, mannitol, sorbitol, etc.) cellulosepreparations and/or calcium phosphates (e.g., tricalcium phosphate,calcium hydrogen phosphate, etc.) as well as binders such as starchpaste using, for example, maize starch, wheat starch, rice starch,potato starch, gelatin, tragacanth, methylcellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/orpolyvinyl pyrrolidine.

Injectable formulations for parenteral administration can be prepared asliquid suspensions, solid forms suitable for solution or suspension inliquid prior to injection, or as emulsions. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol or the like. Inaddition, if desired, the pharmaceutical composition to be administeredmay also contain minor amounts of non-toxic auxiliary agents such aswetting or emulsifying agents, pH buffering agents and the like, such asfor example, sodium acetate, sorbitan monolaurate, triethanolamineoleate, etc.

Aqueous injection suspensions may also contain substances that increasethe viscosity of the suspension, including, for example, sodiumcarboxymethylcellulose, sorbitol, and/or dextran. Optionally, thesuspension may also contain stabilizers.

The parenteral formulations can be present in unit dose or multiple dosesealed containers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, e.g., water, for injections immediately prior touse. Extemporaneous injection suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described.

For oral administration, a pharmaceutically acceptable, non-toxiccomposition is formed by the incorporation of any of the normallyemployed excipients, such as, for example, mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, sodiumcrosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and thelike. Such compositions include suspensions, tablets, dispersibletablets, pills, capsules, powders, sustained release formulations andthe like. Formulations suitable for oral administration can consists ofliquid suspensions such as effective amounts of the drug encapsulatinggagomer particles suspended in diluents such as water, saline, or orangejuice; sachets, lozenges, and troches, each containing a predeterminedamount of the active ingredient as solids or granules; powders,suspensions in an appropriate liquid; and suitable emulsions. Liquidformulations may include diluents such as water and alcohols, e.g.,ethanol, benzyl alcohol, and the polyethylene alcohols, either with orwithout the addition of a pharmaceutically acceptable surfactant,suspending agents, or emulsifying agents.

When the composition is a pill or tablet, it will contain, along withthe active ingredient, a diluent such as lactose, sucrose, dicalciumphosphate, or the like; a lubricant such as magnesium stearate or thelike; and a binder such as starch, gum acacia, gelatin,polyvinylpyrolidine, cellulose and derivatives thereof, and the like.

Tablet forms can include one or more of lactose, sucrose, mannitol, cornstarch, potato starch, alginic acid, microcrystalline cellulose, acacia,gelatin, guar gum, colloidal silicon dioxide, crosscarmellose sodium,talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid,preservatives, flavoring agents, pharmaceutically acceptabledisintegrating agents, moistening agents, and pharmacologicallycompatible carriers.

Capsule forms can be of the ordinary hard- or soft-shelled gelatin typecontaining, for example, surfactants, lubricant, and inert fillers, suchas lactose, sucrose, calcium phosphate, and corn starch.

Lozenge forms can contain the drug encapsulating gagomer particles in acarrier, usually sucrose and acacia or tragacanth, as well as pastillescomprising the active ingredient in an inert base such as gelatin orglycerin, or sucrose and acacia.

In determining the dosages of the gagomer particles to be administered,the dosage and frequency of administration is selected in relation tothe pharmacological properties of the specific active ingredients.Normally, at least three dosage levels should be used. In toxicitystudies in general, the highest dose should reach a toxic level but besublethal for most animals in the group. If possible, the lowest doseshould induce a biologically demonstrable effect. These studies shouldbe performed in parallel for each compound selected.

Additionally, the ED₅₀ (effective dose for 50% of the test population)level of the active ingredient (drug encapsulating gagomer particles) inquestion should be one of the dosage levels selected, and the other twoselected to reach a toxic level. The lowest dose is that dose which doesnot exhibit a biologically demonstrable effect. The toxicology testsshould be repeated using appropriate new doses calculated on the basisof the results obtained.

Young, healthy mice or rats belonging to a well-defined strain are thefirst choice of species, and the first studies generally use thepreferred route of administration. Control groups given a placebo or nottreated are included in the tests. Tests for general toxicity, asoutlined above, should normally be repeated in another non-rodentspecies, e.g., a rabbit or dog. Studies may also be repeated usingalternate routes of administration.

Single dose toxicity tests should be conducted in such a way that signsof acute toxicity are revealed and the mode of death determined. Thedosage to be administered is calculated on the basis of the resultsobtained in the above-mentioned toxicity tests. It may be desired not tocontinue studying all of the initially selected compounds.

Data on single dose toxicity, e.g., LD₅₀, the dosage at which 50% of theexperimental animals die, is to be expressed in units of weight orvolume per kg of body weight and should generally be furnished for atleast two species with different modes of administration. In addition tothe LD₅₀ value in rodents, it is desirable to determine the highesttolerated dose and/or lowest lethal dose for other species, i.e., dogand rabbit.

When a suitable and presumably safe dosage level has been established asoutlined above, studies on the chronic toxicity of the drugencapsulating gagomer particles, its effect on reproduction, andpotential mutagenicity may also be required in order to ensure that thecalculated appropriate dosage range will be safe, also with regard tothese hazards.

Pharmacological animal studies on pharmacokinetics revealing, e.g.,absorption, distribution, biotransformation, and excretion of the activeingredient and metabolites are then performed. Using the resultsobtained, studies on human pharmacology are then designed.

Studies of the pharmacodynamics and pharmacokinetics of the compounds inhumans should be performed in healthy subjects using the routes ofadministration intended for clinical use, and can be repeated inpatients. The dose-response relationship when different doses are given,or when several types of conjugates or combinations of conjugates andfree compounds are given, should be studied in order to elucidate thedose-response relationship (dose vs. plasma concentration vs. effect),the therapeutic range, and the optimum dose interval. Also, studies ontime-effect relationship, e.g., studies into the time-course of theeffect and studies on different organs in order to elucidate the desiredand undesired pharmacological effects of the drug, in particular onother vital organ systems, should be performed.

The compounds of the present invention are then ready for clinicaltrials to compare the efficacy of the compounds to existing therapy. Adose-response relationship to therapeutic effect and side effects can bemore finely established at this point.

The amount of the drug encapsulating gagomer particles of the presentinvention to be administered to any given patient must be determinedempirically, and will differ depending upon the condition of thepatients. Relatively small amounts of the drug encapsulating gagomerparticles can be administered at first, with steadily increasing dosagesif no adverse effects are noted. Of course, the maximum safe toxicitydosage as determined in routine animal toxicity tests should never beexceeded.

Compositions within the scope of the present invention include allcompositions wherein the gagomers encapsulating the water insoluble orpoorly water soluble drug is contained in an amount effective to achieveits intended purpose. While individual needs vary, determination ofoptimal ranges of effective amounts of each compound is within the skillof the art. The dosage administered will depend upon the age, health,and weight of the individual recipient thereof as well as upon thenature of any concurrent treatment and the effect desired. Typicaldosages include 0.01 to 100 mg/kg body weight. The preferred dosages arein the range of about 0.1 to 100 mg/kg body weight. The most preferreddosages are in the range of about 1 to 50 mg/kg body weight.

Preparation of the gagomers with drug entrapment is simple andcost-effective. These drug encapsulating gagomer particles act assustained release drug depots, with half-lives in the range of 19-35hours for the efflux of antibiotics and chemotherapeutics. Theseproperties of the gagomer particles, together with their bioadhesivenature, provide these drug carriers the ability to perform assite-adherent, site-retained, sustained release drug depots forsystemic, including oral, topical, and regional, including intranasal,administrations.

The principles of gagomer preparation are to dissolve the lipid in anorganic solvent and evaporate it to dryness in a manner that forms athin lipid film, which is then hydrated in a basic buffer, usuallyborate buffer at pH9. Alternatively, the lipid can be hydrated directlyin an appropriate basic buffer at a temperature above the lipid's Tm.The glycosaminoglycan is dissolved separately in an acidic aqueous phaseand activated by a water-soluble coupling agent such as a carbodiimide.The hydrated lipid film and the aqueous solution of the activatedglycosaminoglycan are brought together and the system is maintained in abasic pH buffer for the covalent bonding to take place.

Two basic types of gagomers may be synthesized: low lipid toglycosaminoglycan ratio (1:1, w/w), denoted LLG, and high ratio of lipidto glycosaminoglycan (5:1 to 20:1, w/w), denoted HLG. By changingspecific steps in the preparation, the outcome can be directed to formmicro- or nanoparticles.

The gagomers formed by the procedures of the present invention may belyophilized (freeze-dried), with or without drug, and rehydrated withwater alone or rehydrated with an aqueous solution of a drug ofinterest.

Unlike other particulate carriers such as liposomes, there is no need toadd protective agents (cryoprotectants such as sugars) to the gagomersprior to lyophilization, in order to enhance long-term storage andstability of the preparations. The gagomers have intrinsiccryoprotection provided by the hyaluronan (hyaluronic acid).

Following rehydration, the preparation may be heated. Once the gagomershave been lyophilized, they can be stored for extended periods of timeuntil they are to be used. The appropriate temperature for storage willdepend on the lipid formulation of the gagomers and temperaturesensitivity of encapsulated materials.

When the lyophilized gagomers are to be used, rehydration isaccomplished by simply adding an aqueous solution, such as distilledwater or an appropriate buffer, to the gagomers and allowing them torehydrate. This rehydration can be performed at room temperature or atother temperatures appropriate to the composition of the gagomers andtheir internal contents.

The gagomers of the present invention, lipidated glycosaminoglycans, arepreferably prepared by covalently binding a lipid having at least oneprimary amino group, preferably a phospholipid, more preferably aphosphatidylethanolamine, and most preferably dilauryl or dipalmitoylphosphatidylethanolamine, to a carboxylic acid-containingglycosaminoglycan, preferably hyaluronic acid (HA), by the followingmethod:

(1) separately dissolving a lipid and a water insoluble or poorly watersoluble active ingredient in an organic solvent;

(2) combining the dissolved lipid and dissolved water insoluble orpoorly water soluble active ingredient together into a combinedsolution;

(3) evaporating the combined solution to dryness and dispersing as asuspension in a basic borate buffer;

(4) mixing and incubating the dispersed suspension with a solution ofglycosaminoglycan, activated by pre-incubation with a coupling agent, toform lipidated glycosaminoglycan particles encapsulating the waterinsoluble or poorly water soluble active ingredient; and

(5) fractionating by successive centrifugation to enrich for lipidatedglycosaminoglycan particles. The fractionated and enriched lipidatedglycosaminoglycan particles can be further optionally lyophilized.

Alternatively, the lipidated gagomers can be prepared prior toencapsulating the drug/active ingredient by the following method:

(a) A reaction vessel is provided in which the lipid is spread in a thinlayer on the vessel bottom and walls. This can be effected by dissolvingthe lipid in an organic solvent and evaporating the lipid to drynessunder low pressure in a rotary evaporator.

(b) The glycosaminoglycan is activated by pre-incubation in acidic pHwith a crosslinker.

(c) The activated glycosaminoglycan is added to the reaction vessel.

(d) The reaction mixture of the lipid and activated glycosaminoglycan isbuffered to a basic pH in a range of 8.6-9.0.

(e) The buffered reaction mixtures are incubated, with continuousshaking, for a period of time sufficient for the lipidatedglycosaminoglycan to form, such as overnight at 37° C. Since thelipidated gags are designed to be used in vivo, they should be stable atabout 37° C. While higher temperatures can be used for lipidation,lipids undergo physical changes with rising temperatures, generallyabout 62° C. Therefore, the lipidation preferably is conducted attemperatures from about 30-40° C.

(f) The lipidated glycosaminoglycan is buffered to a neutral pH andother ions and water-soluble additives are added according to need inorder to elevate the ionic strength to physiological levels with ions orsalts present in biological fluids (such as NaCl, KCl, Ca²⁺ and Mg²⁺).

(g) The particles are fractionated by successive centrifugations, eachrun at 4° C., for 40 minutes at the g force of 1.6×10⁵, as follows: Thepellet after 3 runs is the microparticle-enriched fraction, thesupernatant of the microparticle enriched fraction subjected to 3additional runs is the nanoparticle-enriched fraction.

(h) The resulting lipidated glycosaminoglycan is lyophilized.

(i) A stock solution of the water insoluble or poorly water solubleactive ingredient is prepared in an organic solvent such as DMSO orethanol. A working solution is then prepared by diluting the stocksolution into water, so that the concentration of the organic solvent is≦1%. This working solution is then used to rehydrate the lyophilizedgagomer powder.

Turbidity studies, following light scattering in a spectrophotometer,may be conducted for equal concentrations of soluble hyaluronic acid andof a gagomer prepared from hyaluronic acid and phosphatidylethanolamineto gain insight into whether the synthesis actually yields particulatematter. As expected, over the concentration range tested free hyaluronicacid is soluble, and its solutions do not scatter light. In contrast,the gagomer-containing samples are turbid, the light scatteringincreasing with the gagomer concentration, making it clear that thebiopolymer is an insoluble material.

The lipidated glycosaminoglycan particles are preferably made withoutany encapsulated materials and then lyophilized to form a powder. Thepowdered glycosaminoglycan particles are then mixed with a powder of thematerial to be encapsulated. Alternatively, the powderedglycosaminoglycan particles are reconstituted by mixing with a solutionof the material to be encapsulated in an organic solvent, which organicsolvent is preferably ethanol. Once the mixture is reconstituted, theparticles will have captured the material that was mixed in. Thus, smallwater-insoluble or poorly water soluble molecules, such as antibioticsand chemotherapeutic drugs, as well as large molecules, can beencapsulated with this technique.

The particles of the present invention are prepared by reacting at leastone glycosaminoglycan in the long form, i.e., the gag has not beensliced up into smaller sizes. All glycosaminoglycans, except hyaluronicacid, are naturally in the form of a protein moiety bound covalently toa poly-saccharide moiety. Methods for hydrolyzing the protein-sugarbond, both chemically and enzymatically, are well known to those skilledin the art. In addition, some commercial products are available in whichthe protein moiety has already been removed.

The glycosaminoglycan polymer is reacted with a lipid which has at leastone primary amino group to cross-link the carboxylic residue of theglycosaminoglycan to a primary amine in the lipid. Once this reactionoccurs, thermodynamic stability causes the lipids to interact with oneanother so as to pull the product into a sphere having theglycosaminoglycan on the outside and the lipids on the inside.Self-assembly of the lipid molecules is a critical force in obtainingthe gagomer particles. These particles are used to encapsulate the waterinsoluble or poorly water soluble drugs/active ingredients in theinterior of the particles.

In one embodiment of the present invention, the protein part of theglycosaminoglycan is removed and only the sugar backbone is reacted withthe lipids.

It is known in the art to attach hyaluronic acid to the outside ofliposomes for targeting or for making the liposomes more bioadhesive. Inthe instant invention, there is no liposome; rather, lipid molecules areattached covalently to hyaluronic acid.

In another embodiment of the present invention, other molecules may beattached first to the glycosaminoglycan, which is then reacted withlipids. The particles produced have these other molecules appearing onthe outside of the particles. These other molecules may be, for example,antibodies, folate, porphyrins, or lectins, and may be used fortargeting.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration and are not intended to be limiting ofthe present invention.

Example 1 Preparation of Paclitaxel-Loaded Gagomers Initial DrugDissolution in DMSO, Followed by Extensive Dilution into Water (Referredto as the Via-DMSO Method)

Lyophilized drug-free gagomer powders were prepared as follows:

Steps (1) and (2) Below were Run in Parallel:

(1) Hyaluronan (HA) was dissolved in water, to a concentration of 2mg/ml. EDC was added to the solution at a ratio of 20 mg/mg HA, the pHwas adjusted to 4.0 by titration with HCl (1M), and the system wasincubated for 2 hours, at 37° C., in a shaker.

(2) 60 mg of dipalmitoyl phosphatidylethanolamine (DPPE) were dissolvedin chloroform:methanol (3:1, v/v) to a concentration of 1.5 mg/ml andthe solution was evaporated to complete dryness under low pressure in arotary evaporator, until a dry uniform film was obtained. Thereafter, 10ml 0.1M Borate buffer, pH 9.0, was added to the dried lipid, thesuspension vigorously agitated for several minutes, then incubated for 2hours at 37° C. in a shaker to create a uniform lipid dispersion.

(3) The solution of activated HA and the basic suspension of the PE weremixed in a 1:1 HA:lipid weight ratio, and incubated overnight at 37° C.in a shaker.

(4) The reaction mixture from (3) was centrifuged in a ultra-centrifugefor 40 minutes at a g force of 160,000, and 4° C. The supernatant wasdiscarded, and the pellet was subjected to 4 cycles of washings asfollows: pellet resuspension in phosphate buffered saline (PBS), pH 7.2,recentrifuged under the conditions listed above. The supernatant wasdiscarded, the pellet was resuspended in PBS and so forth. The finalpellet was suspended in PBS to a desired volume (usually that of system(3)).

(5) The gagomer suspension was dispensed into lyophilization minibottles of 1 ml, and lyophilized as follows: 2 hours freezing at −80°C., with lyophilization over-night (ambient temperature, condensertemperature LT−50° C., pressure 0.055 hPa. The gagomer powder was storedfrozen (−18° C.) until use.

In this example and in all subsequent examples, the gagomerconcentration will be defined by its phosphatidylethanolamine (PE)content.

Stock solutions of paclitaxel in DMSO were prepared in the drugconcentration range of 40 mM. Working paclitaxel solutions were preparedby dilution of these stock solutions in water to the concentration rangeof 170-625 μg/ml and final DMSO concentrations were <1%. A given workingsolution was immediately mixed with a selected quantity of the gagomerpowder to final gagomer concentrations in the range of 0.16-5 mg PE/ml.The mixtures were incubated for 2 hours in a shaker bath at 37° C.Paclitaxel was assayed by inclusion of a trace of ³H-paclitaxel in theformulation.

Efficiency of drug encapsulation is defined as the fraction of totaldrug in the system that is encapsulated within the carrier particles.Paclitaxel encapsulation efficiency was determined by the “thermodynamicmethod”, as follows. Paclitaxel-gagomer formulations were subjected toultra high speed centrifugation (40 minutes, 4° C., g force of 175000).The supernatant (which contains unencapsulated drug) was separated fromthe pellet (which contains the drug-encapsulating gagomers). The pelletwas re-suspended in drug-free buffer to the original volume. Thepaclitaxel concentration was determined in the original formulationbefore the separation, in the supernatant and in the resuspended pellet,and these data were used to determine the efficiency of encapsulation.As shown in FIG. 1, paclitaxel encapsulation efficiency in thesepreparations was very high, 96(±2) %, and was not sensitive to gagomerconcentration.

Example 2 Preparation of Paclitaxel-Loaded Gagomers Initial DrugDissolution in Ethanol, Followed by Extensive Dilution into Water(Referred to as the Via-Ethanol Method)

Paclitaxel-gagomer formulations were prepared similar to those describedin Example 1, but with the following distinction: ethanol was used asthe solvent for the stock solution instead of DMSO. Working paclitaxelconcentrations were in the range of 43-170 μg/ml and final ethanolconcentrations were <1%. As shown in FIG. 2, high encapsulationefficiencies, 80(±6) % and 95(±1) % for the high and low taxolconcentrations, respectively, were also obtained by this approach withslight or no dependence on gagomer concentration within the rangetested.

Example 3 Preparation of Paclitaxel-Loaded Gagomers Initial DrugDissolution in Ethanol, Followed by Addition to the Organic PE Solution(Referred to as the Via-PE Method)

In this approach, paclitaxel was introduced in the course of gagomerpreparation, at the stage in which the PE is dissolved in an organicsolvent system. Paclitaxel was dissolved in ethanol and added to the PEsolution in chloroform:methanol 3:1 v/v, at room temperature (i.e., step2 in Example 1 above). From this point on, gagomer preparation proceededas described in Example 1, except that step 5 (lyophilization) wasomitted.

Paclitaxel final concentrations in the formulation were 0.2-1.2 mM. Asshown in FIG. 3, this method also yields high encapsulationefficiencies, 82(±5) %, with the advantage that this approach allowsencapsulating higher drug doses than the approaches of Examples 1 and 2.This latter point is emphasized by the comparison shown in Table 2, forthe systems in which paclitaxel was dissolved in ethanol. Although theencapsulation efficiency is relatively lower than in the “via PE”method, it allows use of much higher drug doses. Consequently, theencapsulated drug dose in the preferred method (i.e., via-PE) is 5 foldhigher.

TABLE 2 Paclitaxel loading stocks Highest Paclitaxel EncapsulationEncapsulated Method of drug Dose used efficiency Paclitaxel doseencapsulation (μg/ml) (%) (μg/ml) Via ethanol 170 96 163 Via PE 1000 84840

Example 4 Relationship of Encapsulation Efficiency and PaclitaxelLoading

Encapsulation efficiencies for all three methods described in Examples1-3 above were compared vs. the loading level (paclitaxel/PE, wt/wt). Asseen in FIG. 4, there is good agreement between the three encapsulationmethods over a wide range of paclitaxel loadings.

Example 5 Stability in Serum

A comprehensive stability profile of paclitaxel-gagomer formulations inserum requires: (1) Retention of particle integrity that can bemonitored by following, independently, the fate of the twocomponents—hyaluronan and PE and (2) retention of the loaded drug withinthe particle.

To evaluate serum stability of the paclitaxel-gagomer formulations,samples were incubated up to 24 hours in 50% human serum, at 37° C. Togain more insight into the serum effects on particle integrity and itssensitivity to the presence of paclitaxel, similar studies wereconducted with drug-free gagomers. Aliquots were withdrawn from thereaction mixtures at selected time points within the entire duration ofthe experiment, and subjected to separation by centrifugation.Centrifugation conditions and details were similar to those disclosed inExample 1 for the thermodynamic method of determining encapsulationefficiency.

The pellets containing intact gagomers and their encapsulated drug wereresuspended in PBS. Hyaluronan (HA), PE and paclitaxel were assayed inthe withdrawn aliquot before centrifugation, in the supernatant and inthe resuspended pellet, making use of the following trace labels:FITC-labeled HA (F-HA), ¹⁴C-PE, and, where relevant, ³H-paclitaxel. Theresults are summarized in FIG. 5.

The data show quite clearly, for both drug-free and drug-loaded testsystems, that the majority of hyaluronan, independently of PE, areretained in the pellet. This retention is furthermore at the same levelfor both components over most of the time span. These findings allow theconclusion that there is good particle stability in serum. Similarretention of the encapsulated drug further substantiates thisconclusion.

Taking all the data together makes it clear that not only are thegagomers stable in serum, the encapsulated drug is not lost from intactparticles, and its presence does not impair particle stability in serum.

Example 6 Paclitaxel Efflux from Intact Gagomers

The efflux of paclitaxel from the gagomers was studied in both PBS andin 50% human serum under unidirectional flux (sink) conditions, usingthe dialysis approach essentially as described in WO 03/015755 andMargalit et al. (1991). A trace of ³H-paclitaxel was included in thepreparation to assay paclitaxel that was released from the dialysis sacat each time point, as well as the paclitaxel in the gagomers at time=0and at the end of the experiment. Data processing and analysis wasperformed according to the procedures previously developed in thelaboratory of the present inventors (WO 03/015755; Margalit et al.,1991) and found to fit the case of two independent drug pools at time=0,one of encapsulated drug, the other of un-encapsulated drug. The resultsare shown in FIG. 6 and Table 3. This type of data analysis reconstructsthe distribution of drug between the gagomers and the external medium attime=0, and constitutes therefore another approach to determiningencapsulation efficiency, independent of the thermodynamic approachdisclosed in Example 1.

TABLE 3 Parameters of paclitaxel efflux from gagomers in PBS and inserum Gagomer Concen- Efflux of encapsulated tration Paclitaxel (mg f₂⁽²⁾ k₁ k₂ τ_(1/2) PE/ml) Medium (%) (hours⁻¹) (hours⁻¹) (hours) 6 PBS 76± 2 0.54 ± 0.09 0.014 ± 0.002 49 6 Serum⁽¹⁾ 79 ± 2 0.34 ± 0.09 0.006 ±0.002 115 ⁽¹⁾50% human serum in PBS ⁽²⁾Equation (1): f_(t) = (100 −f₂)(1 − e^(−k) ¹ ^(t)) + f₂(1 − e^(−k) ² ^(t)) f_(t)—fraction of totaldrug released from the formulation at time = t f₂: fraction ofgagomer-encapsulated drug at time = 0 k₁: rate constant of efflux forthe unencapsulated drug k₂: rate constant of efflux for the encapsulateddrug

Each data set in FIG. 6 shows the combined accumulation of bothencapsulated and un-encapsulated drug in the dialysates, and it isclearly seen that efflux in the presence of serum is slower than in PBS.As can be seen in Table 3, concomitant with the data and conclusions ofExample 4 above, high serum stability is retained—the f₂ (theencapsulation level) values are quite similar in serum and in PBS. Inboth media, the paclitaxel-gagomer formulations perform assustained-release drug depots, a trait highly-desirable in drugcarriers.

Interestingly, both rate constants are reduced when serum is presentwithin the dialysis sac. Serum components most relevant for the presentcase—efflux of a lipophilic drug—are albumin and lipoproteins, both ofwhich remain within the sac throughout the experiment (the membranecutoff is 12000-14000 Da). As discussed in detail in Margalit et al.(1991), efflux of unencapsulated drug and of the encapsulated drug fromthe dialysis sac to the dialysate is each an independent multi-stepprocess with a single rate limiting step, in which the drug diffusesfrom its original pool through a series of intermediate pool, ending inthe dialysate. This same pattern—a single rate limiting step fordiffusion from each original drug pool—also holds in the present casesof paclitaxel, and the serum vs. PBS differences of the rate constantsimply minor changes in the environment of the rate-limiting drug pools.

Example 7 Cytotoxicity In Vitro

Cytotoxicity of paclitaxel and of gagomer-encapsulated paclitaxel wasevaluated over a matrix of drug-gagomer formulations (see Examples 1, 2and 3), cell lines and treatment protocols, for the drug concentrationrange spanning from 1 nM to 100 μM.

Twenty four hours prior to an experiment, cells of the desired line wereseeded onto 96-well multiwell culture plates at densities in the rangeof 5×10³ cells/well. The experiments were initiated on sub-confluentmonolayers. Upon initiation of an experiment, treatment media was added,as listed in Table 4. Two protocols were used:

(I) Short protocol. The cells were incubated with the treatment mediafor 4 hours, at the end of which the treatment media was aspirated, thecells washed and incubated for 44 hours in drug-free serum-supplementedcell growth media. Termination was 48 hours from start.

(II) Long protocol: the cells were incubated in the treatment media for48 hours. Upon termination, in either protocol, cell viability wasdetermined in each well using either the MTT or the SRB assay method.

TABLE 4 Composition of treatment media for in vitro evaluation ofpaclitaxel-gagomer cytotoxicity. Treatment group Composition oftreatment media 1 Drug-free serum-supplemented cell growth media 2 Freepaclitaxel diluted in serum-supplemented cell growth media 3Paclitaxel-gagomers suspended in serum-supplemented cell growth media 4Drug-free gagomers suspended in serum-supplemented cell growth media

The rationale for the two protocols, the long and the short, is asfollows: The long protocol is the traditional one used for in vitrocomparisons of cytotoxicity among different drug formulations. The cellsare incubated with the treatment formulations for periods usually withinthe 24-72 hour time span, and all formulations are incubated for thesame time. When the comparison is between free drug andcarrier-formulated drug, especially for carriers that have specificpositive interactions with the target cells, this same-incubation periodskews the results in favor of the free drug. In vivo, the free drug willnot remain for long in the target zone, whereas a targeted carrier maystay for prolonged periods. The short protocol is designed to modulatethis free drug vs. carrier-drug imbalance towards the in vivo situation.Removal of the treatment media after 4 hours clears all extracellularfree drug from the system, whereas the drug-loaded carriers canassociated with the cells (bound to the membrane and/or endocytosed). Ifsuch associations take place, not all carrier-mediated drug is clearedupon removal of the treatment media at 4 hours and the remainingdrug-carrier formulations can continue to supply the cells with drugthroughout the remainder of the experiment.

For each cell line, it was verified that the gagomer concentrations used(there was no need to go above gagomer concentration range of 1.5 mgPE/ml) were not toxic to the cells. The data of cell viability as afunction of paclitaxel concentrations were used to extract the IC₅₀(drug concentration causing 50% inhibition of cell proliferation). Thisparameter is in wide use as the field-standard parameter potency—thelower the IC₅₀, the higher the potency of a given formulation.

Paclitaxel IC₅₀ values for free and for gagomer-loaded drug are listedin Table 5. In all cases, and for the three different paclitaxel-gagomertypes of formulation, the data show quite clearly that the encapsulatedpaclitaxel remains active. For both the via-DMSO and the via-ethanolformulations there is no significant difference between the free and thecarrier-formulated drug.

TABLE 5 Cytotoxicity of free and gagomer-encapsulated paclitaxel TX-GAGIC₅₀ short protocol⁽¹⁾ IC₅₀ long protocol Formu- (μM paclitaxel) (μMpaclitaxel) lation Free TX- Free TX- Method Cell line Paclitaxel⁽²⁾GAG⁽³⁾ Paclitaxel GAG Via- B16F10.9⁽⁴⁾ 0.2 0.6 0.03 0.04 DMSO D122 0.93.0 0.25 0.25 C-26 3.0 4.0 3.5 5.0 PANC-1 0.3 0.3 0.02 0.04 COS-7 1 10.06 0.12 Via- SNU-251 0.6 0.9 0.25 0.15 Ethanol Via-PE B16F10.9 1.8 0.40.12 0.22 COS-7 >40 20 5 5 ⁽¹⁾Short protocol: 4 hours of incubation withtreatment media, then: aspiration, wash, incubation for additional 44hours in drug-free serum-supplemented cell growth media. Long protocol:48 hours of incubation with the treatment media. ⁽²⁾Free paclitaxeldiluted directly into medium ⁽³⁾TX-GAG: Paclitaxel-encapsulatinggagomers ⁽⁴⁾Cell origin and type: B16F10.9: mouse melanoma, subline ofB16F10, MDR, over-expressing hyaluronan receptors D122: subline of mouselung Lewis carcinoma, MDR, over-expressing hyaluronan receptors C-26:mouse colon carcinoma, MDR, over-expressing hyaluronan receptors PANC-1:human pancreatic adenocarcinoma, MDR, over-expressing hyaluronanreceptors COS-7: Green African monkey kidneys, subline of CV-1.Non-tumor cells and poor-expressers of hyaluronan receptors SNU-251:Human ovarian cancer, no reports found in the literature with respect tohyaluronan receptors or MDR status.

The results with the via-PE formulation, are also listed in Table 5.These results are the most encouraging of the lot. They show quiteclearly that in the short protocol (which is the more relevant one whencomparing free vs. carrier-formulated drug) there is a drop-down in IC₅₀values. Hence, the increase in potency from the free to thegagomer-formulated drug of more than 4 fold. Taking into account theabove-discussed mechanisms of drug supply to the cells, the increase inpotency when formulated in gagomers may be even higher. Moreover, theincrease in the tumor cell line is significantly higher than in thecontrol, non-tumor line.

Example 8 Preparation and Characterization of Paclitaxel-Loaded GagomersInitial Drug Dissolution in Ethanol, Followed by Addition to EthanolicDPPE Solution (Referred to as the Via Ethanolic-DPPE Method)

This approach is similar to that of Example 3, with the followingdifferences:

(1) The lipid, dipalmitoyl phosphatidylethanolamine (DPPE), wasdissolved in ethanol at 55° C., to a concentration of 12 mg PE/ml.

(2) a stock concentration of paclitaxel was dissolved in ethanol andadded to the ethanolic DPPE solution, and the combined DPPE/taxolethanolic solution was kept at 55° C., for 15 additional minutes.

(3) The DPPE/taxol ethanolic solution was evaporated to dryness anddispersed in the basic borate buffer as described in steps 2 of Example1, except that the incubation of this dispersion was at 65° C. From thispoint, the process continued as in Example 3. Efficiency of paclitaxelwas complete (i.e., 100%). Cytotoxicity in B16D10.9 cells was determinedas described in Example 7. The results, shown in the first row of Table6, show that the gagomer-encapsulated paclitaxel retained activity andis more potent (i.e., lower IC₅₀) than free paclitaxel. Ethanolicsolutions of paclitaxel were heated at several temperatures within therange of 40-70° C., under the same conditions used for making thepaclitaxel-encapsulating gagomers in the method of this example, andusing the same cytotoxicity assay, it was verified that, under thepresent conditions, the heating did not impair the drug's cytotoxicactivity.

TABLE 6 Cytotoxicity of free and gagomer-encapsulated paclitaxel,ethanol as the lipid solvent, in B16F10.9 cells. IC₅₀ short protocol⁽¹⁾TX-GAG (μM paclitaxel) Formulation Free TX- Method Paclitaxel⁽²⁾ GAG⁽³⁾Via 1.8 1.1 Ethanolic-DPPE Via 1.8 0.15 Ethanolic-DLPE ⁽¹⁾Shortprotocol: 4 hours of incubation with treatment media, then: aspiration,wash, incubation for additional 44 hours in drug-free serum-supplementedcell growth media. Long protocol: 48 hours of incubation with thetreatment media. ⁽²⁾Free paclitaxel diluted directly into medium⁽³⁾TX-GAG: Paclitaxel-encapsulating gagomers

Example 9 Preparation and Characterization of Paclitaxel-Loaded GagomersInitial Drug Dissolution in Ethanol, Followed by Addition to EthanolicDLPE Solution (Referred to as the Via Ethanolic-DLPE Method)

Gagomer preparation and paclitaxel encapsulation were all similar tothat described Example 8, except for the following:

(1) DPPE, that has two palmitoyl chains (i.e., C16), was replaced byanother PE—DLPE, in which the two chains are dilauryl (i.e., C12).

(2) The temperature for the lipid dissolution in ethanol, for the mixingof the ethanolic lipid solution with the ethanolic paclitaxel solution,and the temperature for the 2 hour incubation for the dispersion of theDLPE and the paclitaxel in the basic borate buffer were all lowered to44° C. All other steps were similar to those of Example 8.

Efficiency of paclitaxel was complete (i.e., 100%). Cytotoxicity, inB16D10.9 cells was determined as described in Example 7. The results,shown in the second row of Table 6, show that the gagomer-encapsulatedpaclitaxel retained activity, and is more potent (i.e., lower IC₅₀) thanthe free paclitaxel. Among all paclitaxel-gagomer formulations made todate, this formulation (see Tables 5 and 6), was the best in terms ofpreparation, encapsulation efficiency and cytotoxicity.

Example 10 Drug Loading Capacity of TX-GAG Particles

TX-GAG particles were prepared, using DLPE as the lipid, according tothe scheme outlined in FIG. 7. Initial drug/lipid mole ratios rangedfrom 1:10 (i.e., 0.1) to 1:2 (i.e., 0.5) TX:lipid, and encapsulationefficiency was determined for each of these preparations, bycentrifugations and washings, as described Example 1.

To evaluate whether the paclitaxel in the centrifuged pellets iscompletely within the gagomer particle, or some of it—especially as thedrug loading is increased—remains as free insoluble drug outside theparticle, the washing of the TX-GAG particles from excess reagents whichis done by high speed centrifugation, (step 5 in FIG. 7) was done asfollows: Each preparation was divided into two parts. One part wascentrifuged and washed, as in the previous examples, withphosphate-buffered saline (PBS) alone. The other part was centrifugedand washed under similar conditions, but the wash media was PBScontaining 0.2% Bovine-serum albumin (BSA). The rationale for using thelatter stems not only from the traditional use of this protein as ageneric “contaminant absorbing protein”, but specifically becausepaclitaxel has high affinity to this protein and serum albumin acts asthe endogenous carrier of the free paclitaxel given to patients in thecommercial formulation currently in use. If the centrifuged pelletscontained drug which did not enter into the gagomer particles and, dueto its insolubility, precipitated in the pellet together with the TX-GAGparticles, the BSA should have dissolved at least some of theprecipitated free drug. This removal of precipitated free paclitaxelfrom pellet into the supernatant would reduce the drug in thecentrifuged pellet, compared to the equivalent system washed with PBSalone.

The results, shown in FIG. 8, make it quite clear that the BSA wash didnot diminish the drug in the pellet, thus indicating that for allloads—all the paclitaxel associated with the gagomers is indeed loadedinside gagomers. Higher BSA concentrations (3%) also did not “wash out”paclitaxel from the TX-GAG particles.

Example 11 Calorimetric Analysis of TX-GAG Particles

TX-GAG particles, with drug/lipid mole ratios ranging from 1:10 to 1:2were prepared as described in Example 10. Drug-free gagomers weresimilarly prepared, omitting the drug. Both types of systems werelyophilized, as described in step 6 of FIG. 7.

Within the temperature range of 230-250° C., crystalline paclitaxel, aswell as paclitaxel lyophilized from aqueous suspensions (denotedhydrated paclitaxel), are known to undergo first melting, thendecomposition. It is further known that the melting is an endothermicprocess and the decomposition is an exothermic process. Both processes,and their successive pattern of first an endothermic peak, followed byan exothermic peak, can be determined and viewed by subjecting thematter to Differential Scanning Calorimetry (DCS). This is exemplifiedby the two scans, one for crystalline, and the other for hydrated,paclitaxel, in the upper part of FIG. 9.

As also shown in the upper part of FIG. 9, drug-free gagomers subjectedto DSC were found to have a small endothermic peak in the region of 100°C., attributed to the lipid, but to undergo no thermal changes in the230-270° C. range where free paclitaxel undergoes, as shown anddiscussed above, massive changes. Mixtures of drug-free gagomers andcrystalline or hydrated paclitaxel, at drug:lipid mole ratio of 1:2,were also subjected to DSC. As exemplified in the lower part of FIG. 9,the independent components in the mixture retained their individualthermal behaviors, showing the lipid endothermic peak at the region of100° C., and the two successive endothermic and exothermic peaks in the230-250° C. range.

In contrast to all of the above, subjecting TX-GAG formulations to DCS,revealed a different thermal behavior for the encapsulated (compared tofree) drug. As also shown in the lower part of FIG. 9, for a drugloading of 1:2 TX:PE, the melting peak of paclitaxel disappeared whereasthe decomposition peak remained. Similar results were obtained withlower drug:lipid loadings.

For all samples, the identification of the decomposition peak was alsodone by Thermogravimetric Analysis (TGA).

All the above results together fit with the findings of Example 10,strongly indicating that a TX-GAG particle with exceptionally high drugloading is obtained.

Example 12 Paclitaxel Stability in Sterilized TX-GAG Particles

Aqueous suspensions of TX-GAG particles were prepared according to thescheme of FIG. 7, up to and including step 5. Aqueous suspensions ofpaclitaxel alone (i.e., free paclitaxel) were also prepared. Sampleswere set aside, from both the TX-GAG and the free paclitaxel, and theremainder of each system was subjected to the following sterilizationprocess: 12 minutes of autoclaving at 120° C. A critical question ofwhether this sterilization process is feasible for the TX-GAGformulations, is retention of drug activity. Therefore, the originalsamples set aside prior to the sterilization process, as well as thesamples that underwent the process, were tested for their cytotoxicity,in B16F10.9 cultures, as described in Example 7. As shown by the resultsillustrated in FIG. 10, and as expected, the sterilization process didnot generate any significant drop in the cytotoxicity of freepaclitaxel. Moreover, this process also did not cause any significantdrop in the cytotoxicity of the gagomer-loaded paclitaxel. In fact, allfour systems were similar in cytotoxicity, the IC₅₀ value in the rangeof 0.30 μM paclitaxel.

These results indicate that the TX-GAG formulation had good stabilityunder the sterilization conditions applied, and further indicate thatthis is a feasible approach to product sterilization.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the inventions following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

All references cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by references.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

REFERENCES

-   R. Margalit, R. Alon, M. Linenberg, I. Rubin, T. J. Roseman, R. W.    Wood. Liposomal drug delivery: thermodynamic and chemical kinetic    considerations. J. Controlled Release 17:285-296 (1991)

1. A lipidated glycosaminoglycan particle, comprising the reactionproduct of at least one glycosaminoglycan with at least one lipid havinga primary amino group and encapsulating a water insoluble or poorlywater soluble active ingredient.
 2. The lipidated glycosaminoglycanparticle of claim 1, wherein the water insoluble or poorly-solubleactive ingredient is a chemotherapeutic agent for treating cancer. 3.The lipidated glycosaminoglycan particle of claim 2, wherein saidchemotherapeutic agent is paclitaxel.
 4. The lipidated glycosaminoglycanparticle of claim 3, wherein said at least one lipid is dilaurylphosphatidyl ethanolamine.
 5. The lipidated glycosaminoglycan particleof claim 1, wherein said at least one glycosaminoglycan is selected fromthe group consisting of hyaluronic acid, keratan sulfate, chondroitinsulfate, heparin sulfate, heparan sulfate, dermatin sulfate, andfragments, salts, and mixtures thereof.
 6. The lipidatedglycosaminoglycan particle of claim 1, wherein said at least oneglycosaminoglycan is hyaluronic acid.
 7. The lipidated glycosaminoglycanparticle of claim 1, wherein the lipid is a phosphatidylethanolamine. 8.The lipidated glycosaminoglycan particle of claim 7, wherein saidphosphatidylethanolamine is dipalmitoyl phosphatidylethanolamine.
 9. Thelipidated glycosaminoglycan particle of claim 7, wherein saidphosphatidylethanolamine is dilauryl phosphatidylethanolamine.
 10. Thelipidated glycosaminoglycan particle of claim 1, wherein the particlesize is in a range of about 2-5 microns.
 11. The lipidatedglycosaminoglycan particle of claim 1, wherein the particle size is in arange of about 50-200 nanometers.
 12. The lipidated glycosaminoglycanparticle of claim 1, wherein the ratio of lipid to glycosaminoglycan isin a range of 1:1 to 20:1 w/w.
 13. The lipidated glycosaminoglycanparticle of claim 1, wherein the ratio of lipid to glycosaminoglycan isin a range of 5:1 to 20:1 w/w.
 14. The lipidated glycosaminoglycanparticle of claim 1, wherein said at least one glycosaminoglycan has amolecular weight within a range of about 1×10⁵ to about 1×10⁷ daltons.15. The lipidated glycosaminoglycan particle of claim 1, wherein theparticle has a shell of glycosaminoglycan on the outside of the particlewith the lipid portion of the particle forming the inside, without thepresence of liposomes.
 16. The lipidated glycosaminoglycan particle ofclaim 15, which is in the form of a sphere.
 17. A pharmaceuticalcomposition, comprising the lipidated glycosaminoglycan particle ofclaim 1 and a pharmaceutically acceptable carrier, diluent, excipient orauxiliary agent.
 18. A method for preparing the lipidatedglycosaminoglycan particle of claim 1, comprising: separately dissolvinga lipid and a water insoluble or poorly water soluble active ingredientin an organic solvent; combining the dissolved lipid and dissolved waterinsoluble or poorly water soluble active ingredient together into acombined solution; evaporating the combined solution to dryness anddispersing as a suspension in a basic borate buffer; mixing andincubating the dispersed suspension with a solution ofglycosaminoglycan, activated by pre-incubation with a coupling agent, toform lipidated glycosaminoglycan particles encapsulating the waterinsoluble or poorly water soluble active ingredient; and fractionatingby successive centrifugation to enrich for lipidated glycosaminoglycanparticles.
 19. The method of claim 18, further comprising lyophilizingthe fractionated and enriched lipidated glycosaminoglycan particles. 20.The method of claim 18, wherein the lipid is a phosphatidylethanolamine.21. The method of claim 20, wherein said phosphatidylethanolamine isselected from the group consisting of dipalmitoylphosphatidylethanolamine and dilauryl phosphatidylethanolamine.
 22. Themethod of claim 18, wherein said organic solvent is ethanol.
 23. Themethod of claim 18, wherein said glycosaminoglycan is hyaluronan. 24.The method of claim 18, wherein said mixed dispersed suspension andactivated glycosaminoglycan solution has a glycosaminoglycan to lipidweight ratio of about 1:1.
 25. The method of claim 18, wherein saidmixed dispersed suspension and activated glycosaminoglycan solution hasan active ingredient to lipid mole ratio in a range of about 1:10 to1:2.
 26. A method for treating a subject suffering from a pathologicalcondition, comprising administering to said subject an effective amountof the water insoluble or poorly water soluble active ingredientencapsulated in the lipidated glycosaminoglycan particle of claim 1 totreat the pathological condition.
 27. The method of claim 26, whereinthe pathological condition is cancer.
 28. The method of claim 27,wherein the cancer is selected from the group consisting of ovariancancer, breast cancer, colon cancer, head cancer, non-small cell lungcarcinoma, and AIDS-associated Kaposi sarcoma.
 29. The method of claim28, wherein the water insoluble or poorly-soluble active ingredient ispaclitaxel.
 30. In a method for treating an indication with a waterinsoluble or poorly water soluble drug that is effective for treatingsaid indication, the improvement wherein said drug is administeredencapsulated in lipidated glycosaminoglycan particles.